One focus of the group is the characterization of the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. An understanding of how cells sense and protect against oxidative stress is important since the reactive oxygen species generated during normal aerobic growth can oxidize and damage all cellular components. Microarray analysis of the E. coli response to hydrogen peroxide revealed that the sufABCDSE genes, which encode proteins implicated in iron-sulfur cluster assembly, are among the genes induced by oxidative stress. In carrying out additional expression studies and phenotypic characterization of suf mutants, we found that the sufABCDSE operon is specifically regulated to synthesize iron-sulfur clusters when iron metabolism is disrupted by iron starvation or oxidative stress. Assays of the purified SufB, C, D, S and E proteins showed that the SufE protein stimulated the cysteine desulfurase activity of the SufS protein and that the SufBCD complex of proteins enhanced this activity even further. Experiments to further elucidate the function of the SufBCD complex are underway. The key regulator of the inducible defenses against hydrogen peroxide in E. coli is the OxyR transcription factor. We discovered that OxyR is both the sensor and transducer of the oxidative stress signal; the oxidized but not the reduced form of the purified regulator can activate transcription in vitro. OxyR is activated by the formation of an intramolecular disulfide bond between C199 and C208 and is deactivated by enzymatic reduction by glutaredoxin 1 together with glutathione. Structural studies showed that formation of the C199-C208 disulfide bond leads to a large conformational change that alters OxyR binding to DNA. Others have suggested that the activity of OxyR is also modulated by reactive nitrogen species. To evaluate the OxyR contribution to the E. coli response to nitrosative stress, we examined the genome-wide transcriptional responses of cells treated with nitrosylated glutathione or the nitric oxide-generator acidified sodium nitrite during aerobic growth. These assays showed that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the global repressor of ferric ion uptake, are major regulators of the response to reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E. coli defenses against superoxide-generating compounds and hydrogen peroxide, respectively, have minor roles. This study led us to propose that the E. coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species. A central regulator of the response to oxidative stress in S. cerevisiae is the Yap1 transcription factor. Upon activation by increased levels of reactive oxygen species, Yap1 rapidly redistributes to the nucleus where it regulates the expression of up to 70 genes. We purified the Yap1 protein and have been carrying out biochemical experiments to characterize this redox-sensitive transcription factor. Mass spectrometric analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of ~15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p; the amino-terminal cysteine rich domain (n-CRD) and the carboxy-terminal cysteine rich domain (c-CRD). These peptides, which are separated by 250 amino acids in the linear sequence, are joined by the C303-C598 and C310-C629 disulfide bonds. NMR spectroscopy was used to determine the high-resolution solution structure of the redox-domain. In the active oxidized form, a nuclear export signal (NES) in the c-CRD is masked by disulfide-bond-mediated interactions with a conserved alpha-helix in the n-CRD. Point mutations that weaken the hydrophobic interactions between the n-CRD alpha-helix and the c-CRD abolished redox-regulated changes in subcellular localization of Yap1. Upon reduction of the disulfide bonds, Yap1 undergoes a change to an unstructured conformation that exposes the NES and allows redistribution to the cytoplasm. These results revealed the structural basis of redox-dependent Yap1 localization and provided a previously unknown mechanism of transcription factor regulation by reversible intramolecular disulfide bond formation. Another focus of the group is the identification and characterization of noncoding RNAs. Noncoding RNAs are of interest because there is accumulating evidence that many act as important regulators in the cell. Noncoding RNA genes have been missed by most genome annotation, they are usually poor targets in genetic screens and have been difficult to detect by direct sequence inspection. Thus we have been carrying out systematic screens for additional noncoding RNA genes in E. coli. These screens are all applicable to other organisms. One approach based on computer searches of intergenic regions for extended regions of conservation among closely related species has led to the identification of 17 conserved noncoding RNAs. Another screen for noncoding RNAs that coimmunoprecipitate with the RNA binding protein Hfq allowed us to detect six less well conserved RNAs. A third approach of size fraction of total RNA followed by linker ligation and cDNA synthesis has led to the cloning of cis-encoded antisense RNAs. We previously showed that OxyS RNA, whose expression is induced by OxyR in response to oxidative stress, acts to repress translation by basepairing with target mRNAs. OxyS RNA action is dependent on the Hfq protein, which functions as a chaperone to facilitate OxyS RNA basepairing with its target mRNAs. We also discovered that the abundant 6S RNA binds and modifies RNA polymerase. In the last year, we have elucidated the functions of two recently-discovered noncoding RNAs that bind Hfq: the 109 nucleotide MicC RNA and the 105 nucleotide GadY RNA. We found that MicC represses translation of the OmpC outer membrane porin. Interestingly, under most conditions, the MicC RNA shows the opposite expression as the MicF RNA, which represses expression of the OmpF porin. Thus we suggest that the MicF and MicC RNAs act to control the OmpF:OmpC protein ratio in response to a variety of environmental stimuli. In contrast, basepairing between the GadY RNA and the 3?-untranslated region (3? UTR) of the gadX mRNA encoded opposite gadY leads to increased stability of the gadX mRNA. Enhanced gadX mRNA stability results in increased GadX levels and increased expression of the acid-response genes controlled by the GadX transcription factor. Studies to further characterize the GadY RNA activity and the roles of other newly-discovered noncoding RNAs are ongoing.